The nuclear orphan receptor CAR (constitutively active receptor or constitutive androstane receptor) activates transcription of genes encoding various drug-metabolizing enzymes. For example, CAR activates xenochemical/steroid-metabolizing enzymes, in response to phenobarbital (PB) and other PB-type inducers (Sueyoshi and Negishi, Annu Rev Pharmacol Toxicol 41:123-43, 2001; Wei et al., Nature 407:920-3, 2000; Zelko and Negishi, Biochem Biophys Res Commun 277:1-6, 2000; and Ueda et al., Mol Pharmacol 61:1-6, 2002). Induction of these enzymes confers on organisms a higher metabolic capability to defend themselves against xenochemical toxicity and/or carcinogenicity.
In addition to xenochemicals such as PB and TCPOBOP (1,4-bis [2-(3,5-dichloropyridyloxy)]benzene), steroid hormones can modulate the CAR-mediated activation of gene transcription. Estrogens activate mouse CAR (mCAR) and induce the Cyp2b10 gene in mouse liver, whereas androgens and progesterone repress estrogen-activated mCAR (Kawamoto et al., Mol Endocrinol 14:1897-1905, 2000). However, unlike mCAR, human CAR (hCAR) does not respond to steroid hormones (Id.).
Direct activation of CAR in response to various drugs has been observed in vivo. CAR is retained in the cytoplasm of non-induced mouse livers and accumulates in the nucleus following PB treatment (Kawamoto et al., Mol Cell Biol 19:6318-22, 1999). However, in vitro, CAR is active even in the absence of agonistic chemicals in a cell-based transfection assay (Kawamoto et al., Mol Endocrinol 14:1897-1905, 2000). This constitutive activity makes it difficult to use CAR in vitro to screen for drugs that can enhance activation of xenochemical/steroid metabolizing enzymes. As a result, there have been attempts to alter CAR's in vitro constitutive activity.
Some steroid metabolites, such as androstenol, repress the constitutive activity of CAR in a cell-based transfection assay (Forman et al., Nature 395:612-5, 1998). In addition, PB and PB-type inducers can re-activate the repressed CAR in vitro (Sueyoshi et al., J Biol Chem 274:6043-6, 1999). Additional repressors such as progesterone, androgens, and CaMK inhibitor KN-62, as well as activators, such as estrogens, have been identified (Kawamoto et al., Mol Endocrinol 14:1897-1905, 2000). Thus, the re-activation is one method that can be used to regulate CAR in xenochemical-responsive fashion, at least, in vitro. However, whether this is also the regulatory mechanism in liver in vivo remains unclear.
It would be beneficial if the constitutively active nature of CAR could be converted into a xenochemical-responsive one, so that screening for drugs that can enhance activation of xenochemical/steroid metabolizing enzymes could also be readily performed in vitro, instead of only in vivo.